RNA-sequencing analysis of NGN3-GFP positive and negative cells purified from differentiating human embryonic stem cells

NGN3-eGFP could be used to mark endocrine progenitor cells and their progeny in differentiating hESCs, suggesting that profiling analysis of NGN3-eGFP+ cells could lead to the discovery of novel gene participation. Six batches of NGN3-eGFP+ and NGN3-eGFP -cells purified by FACS from a reporter hES cell line cell cultures at stage 4 were pooled together, respectively. RNA-seq-based analysis of the two cell fractions identified a total of 3976 differentially expressed genes (P-value<0.05; Fold >2), of which 72% are enriched in NGN3-GFP+ cells. As expected, all the genes expressed in pancreatic progenitor cells are enriched in NGN3-eGFP-cells; while all endocrine-associated genes are enriched in NGN3-eGFP+ cells. Among the 3976 genes, 195 have potential transcriptional factor activity and 453 are potential non-coding RNA genes, most of which have not been well investigated in pancreatic endocrine cells. The gene ontology term ‘integral to membrane’ GO: 0016021 identified 1003 differentially expressed genes that encoded ion channels, adhesion molecules and transporters.